Method of producing non-human mammals

ABSTRACT

A method of producing mutant/targeted non-human mammals, such as mutant mice that does not require production of chimera and permits the introduction of multiple mutations in embryos and, thus, avoids the necessity of breeding to combine all of the desired mutations in a single animal. The method is efficient in producing ES mice.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/957,659, filed Sep. 20, 2001, which is a continuation-in-part of U.S.application Ser. No. 09/755,003, filed Jan. 5, 2001, which claims thebenefit of the filing date of U.S. Provisional Application No.60/234,378, filed Sep. 20, 2000 and the filing date of U.S. ProvisionalApplication No. 60/255,970, filed Dec. 15, 2000. This application isalso related to U.S. application Ser. No. 10/787,847, filed Feb. 26,2004. The entire teachings of the referenced applications areincorporated herein by reference.

GOVERNMENT SUPPORT

Work described herein was supported, in whole or in part, by NationalInstitutes of Health Grants No. 5-R35-CA44339 and RO1-CA84198. TheUnited States government has certain rights in the invention.

BACKGROUND OF THE INVENTION

In the past two decades, considerable effort has been invested inproducing non-human mammals, such as mutant or transgenic mammals, suchas mice, and during that time, a variety of methods have been developed.In order to produce a desired mutant animal, such as a mouse, using anyof the presently available methods, one must first produce chimeras andbreed the chimeras to produce homozygous offspring; production ofoffspring which are not chimeric requires two breeding cycles for eachgene. This is the case for each mutation to be introduced and, ifoffspring exhibiting more than one mutation are desired, additionalbreeding cycles are required. For example, if mutant mice bearing sixdifferent alterations (e.g., six different genes) are to be produced,approximate breeding time will be two years. Producing desiredgenetically manipulated mammals, even those for which the breeding cycleis relatively brief, requires considerable time, as well as resources,using current methods. It would be very valuable if a more efficientmethod of producing mutant mammals, such as mice, were available.

SUMMARY OF THE INVENTION

The present invention relates to methods of producing non-human mammals,which can be mutant non-human mammals or non-mutant non-human mammals,such as mice, that do not require production of chimera or chimericoffspring (offspring that consist of cells that are derived from morethan one zygote). The invention is a method of producing non-humanmammals, such as mutant or non-mutant mice, by tetraploid blastocystcomplementation using non-inbred pluripotent cells or cell lines, suchas non-inbred ES cells or cell lines.

The present method makes it possible to include multiple mutations oralterations in the same pluripotent cells (e.g., embryonic stem (ES)cells) or cell lines (e.g., ES cell lines) before producing an animalfrom the ES cells or ES cell lines. The present invention relates tomethods of producing non-human mammals that, thus, avoid thetime-consuming step of breeding chimera to produce the desiredoffspring. As is evident from the work described herein, mutant ortargeted offspring, particularly mice, that are entirely derived from EScells or ES cell lines and survive postnatally have been producedwithout the need to produce chimeric intermediates. Mutations introducedinto the non-inbred pluripotent cells or cell lines can be non-random ortargeted alterations or can be random or non-targeted alterations. Theproducts of either approach are referred to herein as mutant. In thoseembodiments in which mutations are non-random or targeted, the resultingproducts can also be referred to as targeted (e.g., targeted ES cells,targeted ES cell lines, targeted non-human mutant mammals, such astargeted mutant mice). Alterations can be of a variety of types,including deletion, addition, substitution, or modification of all or aportion of DNA (e.g., a gene, regulatory element) in the ES cells. Thesealterations include addition of a gene or gene portion not normallypresent in the ES cells or ES cell lines. Non-mutant mice that arederived entirely from ES cells or ES cell lines and survive postnatallycan also be produced using the method described. The present methods ofproducing mice, particularly mutant mice, make it possible to produceoffspring, particularly mutant offspring, very efficiently, particularlyin comparison with other methods.

The present invention also relates to a method for deriving fertile XOfemale mice from non-inbred (F1) mouse male ES cells or non-inbred (F1)mouse male cell lines and a method of deriving males and femalescarrying all genetic alterations introduced into a single non-inbred ESclone, such as a targeted non-inbred mouse ES cell clone. Breeding ofthe mutant males and females allows the production of a mutant mousestrain derived from a single non-inbred ES cell clone, such as atargeted (e.g., multiply targeted ES cell clone), without outcrossingthe mutant animal with a wildtype partner, as is required in presentlyavailable methods.

The present invention also relates to non-human mammals, particularlymutant non-human mammals, such as mutant mice, produced by the methods;cells obtained from the mutant or non-mutant non-human mammals and celllines produced from these cells. A particular embodiment is cellsobtained from mutant or non-mutant mice produced by a method of thepresent invention; cells obtained from the mice and cell lines producedfrom such cells. The invention further relates to a method of producingblastocysts useful in the method of producing mutant or non-mutantmammals, such as mouse blastocysts (non-mutant or mutant) useful forproducing non-mutant or mutant mice by the method described herein andblastocysts produced by the method.

The invention also relates to methods of identifying XO F1 ES cells(e.g., mouse XO F1 ES cells) comprising screening a population of F1 EScells, such as a population of wildtype or mutant F1 ES cells, for F1 EScells that are XO F1 ES cells. In a particular embodiment, screening iscarried out to identify F1 ES cells in which spontaneous loss of the Ychromosome has occurred, resulting in XO F1 ES cells. By population ofF1 ES cells is meant a mixture of XO F1 ES cells and XY F1 ES cells.

In particular embodiments, mutant non-human mammals (e.g., mutant mice)are produced to mimic or serve as a model for a condition (e.g., aneurological, muscular or respiratory condition, cancer, viralinfection, arthritis,) that occurs in another species, such as inhumans. They are used to identify new drugs that have a therapeutic orpreventive effect on the condition or assess the ability of known drugsto act as therapeutics or preventatives. Thus, the present inventionencompasses methods in which the mutant non-human mammals (particularlymutant mice) are used, such as in a method of screening to identify anew drug that inhibits the occurrence of (prevents the onset, reducesthe extent or severity of) or reverses a condition caused by orassociated with the genetic alteration(s) and a method of screeningknown drugs for those that inhibit onset of or reverse such conditions.Drugs identified by methods in which the mutant mammals of the presentinvention are used are also the subject of this invention. These includedrugs that inhibit onset of a condition (prevent the onset or reduce theextent to which the condition is established or severity of thecondition), referred to as preventatives or prophylactic drugs and drugsthat reverse (partially or completely) or reduce the extent or durationof the condition once it has occurred.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, Applicants have demonstrated that geneticbackground is a crucial parameter controlling postnatal survival ofoffspring that are entirely derived from ES cells or ES cell lines. Thatis, heterozygosity of the genome of the pluripotent donor cell (e.g.,heterozygosity of the donor ES cell genome) is critical for postnatalsurvival of offspring whose development is achieved without thecontribution of normal cells derived from the host embryo. Further,Applicants have demonstrated that non-human mammals, particularly mice,can be generated without the need to first create a chimericintermediate. The ability to derive offspring (e.g., mice) directly fromES cells or ES cell lines without the need to produce chimericintermediates is a distinct advantage, not only because it avoids thetime consuming and expensive step of producing chimera, but also becauseit facilitates the generation of offspring with multiple geneticalterations. The generation of F1 ES cell-tetraploid mice provides asimple procedure for directly deriving animals with complex geneticalterations without the need to create a chimeric intermediate. Thetetraploid technology in combination with the use of F1 cells or F1 celllines allows assembly or production of multiple genetic alterations inthe same ES cell clone by consecutive gene targeting cycles in vitro.The resulting multiply targeted F1 ES cell clone is introduced intotetraploid blastocysts to produce an embryo that is then transferred toan appropriate foster mother and permitted to develop to term. Thus, atransgenic animal with one or multiple desired or selected geneticalterations can be generated without the need for production of chimericfounders and outbreeding with wild type mice.

Also described herein is a strategy for deriving fertile XO females fromF1 male ES cells or F1 male ES cell lines and a method of breeding amutant mouse strain derived from a given multiply targeted ES cell clonewithout outcrossing the mutant animal with a wild type partner. Thisavoids time consuming and costly outcrossing, which would otherwise benecessary. Because each F1 ES cell line is of a given sex—usuallymale—it would not be possible to breed a mutant mouse strain derivedfrom a given multiply targeted ES cell clone without outcrossing, usingpresently available methods. As described herein, however, outcrossingis no longer required, in view of the fact that it is possible togenerate mutant males and females from a single targeted male ES cellclone by selection for loss of one Y chromosome, resulting in generationof XO ES cells. In one embodiment, a negative selection marker (e.g., anegative selection gene, such as a Herpes Tk gene) is introduced intothe Y chromosome of F1 male ES cells, as described further below, andthe resulting cells are subject to selection with an agent (e.g.,gancyclovir) which kills all cells carrying the Y chromosome. Cells thatare not killed have lost the Y chromosome and, thus, are XO. Thisenables subsequent generation of males and females carrying identicalgenetic alterations, as described further below.

The invention described herein relates to a method of producingnon-human mammals, which can be mutant or non-mutant animals, such asmutant or non-mutant mice. As described herein, it has now been shownthat mutant non-human mammals can be produced without the intermediatestep of producing chimeric animals which, in presently availablemethods, must be bred to produce the desired mutants. In particular,targeted or mutant mice have been produced and the present invention isdescribed in detail by describing their production. However, the presentinvention is useful to produce mutants or non-mutants of any non-humanmammal for which embryonic stem (ES) cells can be obtained.

The invention is, in one embodiment, a method of producing a non-humanmammal. The method comprises introducing non-inbred pluripotent cells,such as non-inbred ES cells, into tetraploid blastocysts of the samemammalian species, under conditions that result in production of anembryo (at least one/one or more embryo) and transferring the resultingembryo(s) into an appropriate foster mother, such as a pseudopregnantfemale of the same mammalian species. The resulting female is maintainedunder conditions that result in development of live offspring, therebyproducing a mutant non-human mammal. The resulting non-human mammal isderived from a single zygote (that which originally gave rise to the EScells). Such mammals are referred to herein as ES non-human mammals.

In another embodiment, the invention is a method of producing a mutantnon-human mammal. The method comprises introducing non-inbredpluripotent cells, such as non-inbred ES cells, comprising at least onemutation or alteration into tetraploid blastocysts of the same mammalianspecies, under conditions that result in production of an embryo (atleast one/one or more embryo) and transferring the resulting embryo(s)into an appropriate foster mother, such as a pseudopregnant female ofthe same mammalian species. The resulting female is maintained underconditions that result in development of live offspring, therebyproducing a mutant non-human mammal. The resulting mutant non-humanmammal is derived from a single zygote (that which originally gave riseto the ES cells). Such mammals are referred to herein as mutant ESnon-human mammals. The mutations or alterations can be non-random ortargeted or, alternatively, can be introduced randomly or in anon-targeted manner.

A specific embodiment of the present invention is a method of producinga targeted or mutant mouse, comprising: (a) introducing mouse non-inbredpluripotent cells comprising at least one alteration in genomic DNA intomouse blastocysts, preferably tetraploid blastocysts, thereby producingmouse blastocysts containing mouse non-inbred pluripotent cells; (b)maintaining the product of (a) under conditions that result inproduction of embryos; (c) introducing an embryo or embryos (at leastone/one or more embryos) into a foster mother, such as a pseudopregnantfemale mouse; and (d) maintaining the female into which the embryo(s)were introduced under conditions that result in development of liveoffspring, thereby producing a mutant mouse. The mutant mouse is alsoreferred to herein as a mutant ES mouse. In one embodiment, thenon-inbred mouse pluripotent cells are non-inbred mouse ES cells, suchas F1 cells derived from two different strains of mice or F2, F3 F4,etc. cells that can be derived from parents after consecutivebrother-sister matings. Alternatively, such cells can be derived fromparents after backcrossing an F1 strain to one of the parent strain toobtain the first backcross generation (N1) and by further backcrossingto obtain N2, N3, N4, etc. backcross generations. As used herein, theterm non-inbred ES cells encompasses all of the hereinabove described EScells and cell lines. Derivation of non-inbred ES cells, with specificreference to production of mouse ES cells, is described in detail in theExamples.

In a further embodiment, the invention is a method of producing anon-mutant mouse, referred to as a non-mutant ES mouse. The method iscarried out as described above for the production of mutant ES mice,except that DNA in the non-inbred pluripotent cells, such as non-inbredES cells (e.g., non-inbred mouse cells) has not been altered prior totheir use. That is, the non-inbred ES cells as obtained may containalterations or mutations, but are not further modified to producenon-random or random mutations. The method comprises: (a) introducingmouse non-inbred pluripotent cells into mouse blastocysts, preferablytetraploid blastocysts, thereby producing mouse blastocysts containingmouse non-inbred pluripotent cells; (b) maintaining the product of (a)under conditions that result in production of embryos; (c) introducingan embryo or embryos (at least one/one or more embryos) into a fostermother, such as a pseudopregnant female mouse; and (d) maintaining thefemale into which the embryo(s) were introduced under conditions thatresult in development of live offspring, thereby producing a non-mutantmouse.

A variety of methods can be used to introduce mouse pluripotent cells,such as non-inbred mouse ES cells, into mouse tetraploid blastocysts. Inone embodiment, this is carried out by injecting the non-inbred cellsinto tetraploid blastocysts, such as by microinjection, particularlypiezo microinjection. Other methods can be used to introduce non-inbredES cells into the blastocysts. For example, the method described byAmano et al., or a modification thereof, can be used (Amano, T. et al.,Theriogenology 53, 1449-1458 (2000)). Alternatively, any other method,such as a chemical method, which results in introduction of non-inbredES cells into tetraploid blastocysts can be used.

Non-inbred pluripotent cells, such as non-inbred ES cells, used in thepresent method can contain at least one/one or more genetic alterationsor mutations. Alternatively, as described above, non-inbred ES cellsused can be non-mutant (have not been altered, after they are obtained,to contain a genetic alteration or mutation); such cells are used toproduce non-mutant progeny by the method of the present invention. Thegenetic alterations or mutations that can be present in non-inbred EScells used include, but are not limited to, transgenes (cDNA, genes orportions thereof), mutations (targeted or random), conditionalmutations, targeted insertions of foreign genes, YAC and BAC sizedtransgenes, all or part of a chromosome, which may be from the samespecies as the embryo or another species, such as from a human. Theyinclude physical knockout of all or a part of a gene, functionalknockout of a gene, introduction of a functional gene and introductionof DNA or a gene portion that changes the function/level of expressionof a gene present in the ES cell (e.g., a promoter, enhancer orrepressor). An important feature of the method of the present inventionis that multiple genetic alterations, which will typically beconsecutive genetic alterations but can also be simultaneous, can bemade in the non-inbred ES cells, thus circumventing the need forbreeding to combine multiple alterations in one animal, as is requiredif presently-available methods are used. Alterations can also be presentin the non-inbred ES cells as they are obtained from the zygote fromwhich they are derived. As used herein, the terms mutant non-inbredpluripotent cells, mutant non-inbred ES cells and similar termsencompass cells which comprise a mutation or mutations as obtained fromthe zygote which gave rise to the cells and cells which are mutated oraltered after they are obtained from the zygote. Alterations can all beof the same type (e.g., all introduction of exogenous DNA) or of morethan one type (e.g., introduction of exogenous DNA, gene knockout andconditional gene knockout). They can also be a combination of mutationspresent in the non-inbred ES cells as derived from a zygote andmutations made after they are derived from a zygote. The alterationsmade in genomic DNA of non-inbred ES cells can be chosen to produce aphenotype that is similar to (mimics) a condition that occurs in otherspecies (e.g., humans) and the resulting mutant mice can, thus, serve asa model for that condition.

A variety of methods, known to those of skill in the art, can be used toalter or mutate inbred pluripotent (e.g., ES) cells or cell lines to beused in the method of producing ES mice of the present invention. Forexample, an appropriate vector or plasmid can be used to introduce DNAinto ES cells in order, for example, to integrate DNA into genomic DNA,express foreign DNA in recipient cells, cause recombination (homologousor nonhomologous) between introduced DNA and endogenous DNA or knock outendogenous gene(s), such as by means of the Cre-lox method.Alternatively, alterations or mutations can be produced by chemicalmethods or radiation. Gene targeting can also be used to produce mutantnon-inbred pluripotent cells or cell lines, such as mutant non-inbred EScells or cell lines. For example, the methodology described by Rideoutand co-workers can be used. See, Rideout, W.M. et al., Nature Genetics,24:109 (2000) and the references cited therein.

Tetraploid blastocysts can be produced by known methods, such as thatdescribed by James and co-workers. James, R. M. et al, Genet. Res.Camb., 60:185 (1992). See also Wang, Z-Q et al, Mech. Dev., 62: 137(1997) and the references cited therein.

The invention also relates to methods of identifying XO F1 ES cells,such as mouse XO F1 ES cells, by screening a population of F1 ES cells,such as a population of wildtype or mutant F1 ES cells, for F1 ES cellsthat are XO F1 ES cells. By population of F1 ES cells is meant a mixtureof XO F1 ES cells and XY F1 ES cells. In a particular embodiment,screening is carried out to identify F1 ES cells in which spontaneousloss of the Y chromosome has occurred, resulting in XO F1 ES cells. Theinvention further provides methods for isolating XO F1 ES cells from XYF1 ES cells, comprising (a) screening a population of F1 ES cells (e.g.,wildtype or mutant) for loss of the Y chromosome; and (b) isolating F1ES cells lacking the Y chromosome, thereby isolating XO F1 ES cells.Screening can be carried out using a variety of methods known andreadily available in the art, such as using a Y chromosome probe (e.g.,against repetitive elements) in Southern blot analysis or PCRamplification. F1 ES cells can be generated using a variety of methodsknown and readily available in the art, such as, for example, bylimiting dilution subcloning or transfection with exogenous DNA followedby appropriate selection. By exogenous DNA is meant any DNA that is notendogenous to the cells to be transfected or that does not already occurin the cells to be transfected. An example of an exogenous DNA is a drugselection marker.

Also the subject of this invention are mutant non-human mammals (e.g.,mutant ES mice) produced by the method described herein; methods ofproducing non-human mammalian embryos; non-human embryos produced by themethod; and a method of identifying a drug to be administered to treat acondition that occurs in a mammal, such as a human. The method ofproducing mutant non-human mammalian embryos comprises injectingnon-human F1 ES cells into non-human tetraploid blastocysts andmaintaining the resulting tetraploid blastocysts under conditions thatresult in formation of embryos, thereby producing a mutant non-humanmammalian embryo(s). In one embodiment, the non-human mammalian embryois a mutant mouse embryo.

Another embodiment of the present invention is a method of producing anon-human mammalian strain, such as a mouse strain, particularly amutant mouse strain, that is derived from a given (single) ES cellclone, such as a mutant non-inbred ES cell clone, without outcrossingwith a wildtype partner. Until now, it has not been possible to do sobecause each F1 ES cell line is of a given sex (generally male).However, fertile XO females have been produced from a male ES cell cloneby in vitro selection for loss of the Y chromosome. As a result, amutant mouse strain carrying all genetic alterations can be derived bybreeding XY males and XO females, both derived from the same targeted EScell clone and of identical genetic makeup without outbreeding of themutant male to a normal female. Production of XY and XO subclones fromthe same clone, such as a clone carrying one or more genetic mutation ora multiply targeted clone, makes it possible to generate males andfemales carrying the same genetic alterations; such males and femalescan then be used to produce genetically identical offspring without theneed for outbreeding.

For example, insertion of a negative selection marker (e.g., the HerpesTk gene) on the Y chromosome of F1 ES cells makes it possible to deriveXY and XO subclones from an initial male ES cell clone. The XO ES cellscan be produced, for example, by inserting a Herpes Tk gene onto the Ychromosome by homologous recombination using known methods. For example,a vector that contains sequences homologous to a Y-linked gene (such asthe Sry gene, the Mov15 gene or any other Y-linked gene) and expressesthe Tk gene can be produced and introduced into ES cells. The cells aremaintained under conditions that result in homologous recombinationbetween vector sequences and Y chromosome sequences. The Tk gene is, asa result, introduced into the Y chromosome. The resulting cells can thenbe targeted or otherwise genetically altered. To generate an XO linefrom the clones, the cells are subjected to selection by culturing inthe presence of gancyclovir, which results in killing of all cellscarrying the Y chromosome into which the Tk gene has been inserted. XOcells can be used to produce XO females, using known methods. Similarly,XY cells can be used to produce males. The resulting males and femalescan be bred to produce offspring carrying the same genetic material(mutations) as the parents. Other negative selection markers, such asdiphtheria toxin, can be used and are introduced into the Y chromosomein a manner similar to that described for introduction of the Tk gene.Alternatively, XO ES cells can be produced by introducing DNA into XY EScells, such as by homologous recombination to functionally or physicallydelete the Y chromosome. The desired XO cells can be identified, forexample, by use of a Y chromosome probe (e.g., for repetitive elements)in Southern blot analysis. In addition, because the Y chromosome isfrequently lost spontaneously upon in vitro culture of ES cells, themutant male F1 ES cells can be passaged and subclones screened forspontaneous loss of the Y chromosome. XO ES cells can be identified, asdescribed in Example 3, for example, through Southern blot analysis inwhich a Y chromosome probe against repetitive elements is used. XO EScells can also be identified using other methods known and readilyavailable in the art, such as, for example, by PCR. Fertile female micehave been produced from male F1 ES cells that have a male karyotype andare positive for Y-specific sequences by PCR in early passages. TheB6×Balb line, V30.11 has produced 4/4 females who, as judged, forexample, by their coat color, should be totally derived from male EScells.

The method of the present invention is, thus, also a method of producinga mutant mouse by breeding a mutant male mouse and a mutant femalemouse, wherein the male mouse (or an ancestor thereof) and the femalemouse (or an ancestor thereof) were produced from the same F1 male EScells or cell line, such as from the same targeted male ES cell clone,and the female mouse is an XO female. That is, the method is one ofproducing a mutant mouse strain by breeding a mutant male mouse and amutant female mouse carrying identical genetic alterations as a resultof having been derived from a single targeted male F1 ES cell clone. Themutant female mouse is XO and is produced (or is the progeny of anancestor which was produced) by selecting for loss of the Y chromosomefrom a single (individual) male ES cell clone. The present inventionalso encompasses female mice which are XO and were produced from an F1male ES cell or cell line, such as by knocking out of the Y chromosome.It also encompasses progeny produced by breeding a male and a femaleproduced as described herein and progeny thereof.

The mutant non-human mammals, such as mutant mice, can be used as amodel for a condition for which a preventive or therapeutic drug issought. A method of identifying a drug to be administered to treat acondition in a mammal comprises producing, using the method of thepresent invention, a mutant mouse that is a model of the condition;administering to the mutant mouse a drug, referred to as a candidatedrug, to be assessed for its effectiveness in treating or preventing thecondition; and assessing the ability of the drug to treat or prevent thecondition. If the candidate drug reduces the extent to which thecondition is present or progresses or causes the condition to reverse(partially or totally), the candidate drug is a drug to be administeredto treat the condition.

The present invention is illustrated by the following examples, whichare not intended to be limiting in any way.

EXAMPLES

The following examples describe production of mice using inbred ES cellsfrom four different ES cell lines from three inbred backgrounds (129/Sv,C57BL/6 and BALB/c) and six different F1 lines (129/Sv×C57BL/6,C57BL×129/Sv, BALB/c×129/Sv, 129/Sv×M. castaneus, C57BL/6×BALB/c and129/Sv×FVB); assessment of pups produced using the two types of EScells; and comparison of results obtained. The results show that use ofF1 ES cells consistently results in production of viable mice, whethertargeted or untargeted cells are used. They also demonstrate thatgenetic background is a crucial parameter for postnatal survival of pupsderived from ES cells. Further, they demonstrate that the method of thepresent invention has been successfully used to produce mice thatcontain desired alterations without the need to produce and breedchimera en route to producing the desired non-chimeric pups.

Methods and Materials

The following methods and materials were used to produce mouse pups.

Production of ES Cell Clones

Nuclear transfer of ES cell nuclei into enucleated metaphase II oocyteswas carried out as previously described (Wakayama, T. et al., Nature,394:369-374 (1998); Wakayama, T. & Yanagimachi, R., Nature Genet.,22:127-128 (1999); Ogura, A. et al., Biol. Reprod., 62:1579-1584 (2000);Rideout, W. M. et al., Nature Genet., 24:109-110 (2000);Wakayama, T. etal., Proc. Natl. Acad. Sci. USA, 96: 14984-14989 (1999)). 1-3 hoursafter nuclear transfer oocytes were activated for 5 hours with 10 mMSr⁺⁺ in Ca⁺⁺ free media in the presence of 5 mg/ml of Cytochalasin B.Embryos were cultured in vitro to the blastocyst stage and transferredto recipient mothers.

Embryo Culture

All embryo culture was carried out in microdrops on standard bacterialpetri dishes (Falcon) under mineral oil (Squibb). Modified CZB media(Chatot, C. L. et al., Biol. Reprod., 42: 432-440 (1990)) was used forembryo culture unless otherwise noted. Hepes buffered CZB was used forroom temperature operations while long term culture was carried out inbicarbonate buffered CZB at 37° C. with an atmosphere of 5% CO₂ in air.

Recipient Females and Cesarean Section

Ten injected blastocysts were transferred to each uterine horn of 2.5days post coitum pseudopregnant Swiss females that had mated withvesectomized males. Recipient mothers were sacrificed at E 19.5 and pupswere quickly removed from the uterus. After cleaning fluid from theirair passages, pups were placed under a warming light and respiration wasobserved. Surviving pups were fostered to lactating BALB/c albinomothers.

Culture of Embryonic Stem (ES) Cells

Derivation and culture of embryonic stem cells were carried out aspreviously described (Nagy, A. et al., Development, 110:815-821 (1990))with ES cell lines derived from both inbred and F1 blastocysts. ES cellswere cultured in DMEM with 15% FCS containing 1000 U/ml LeukocyteInhibiting Factor (LIF) on gamma-irradiated primary feeder fibroblasts.For blastocyst injection ES cells were trypsinized, resuspended in DMEMand preplated on a standard 10 cm tissue culture dish for thirty minutesto remove feeder cells and debris.

Preparation of Two Cell Embryos for Electrofusion B6D2F1 females weresuperovulated by IP injection of 7.5 IU PMS (Calbiochem) followed 46-50hours later with 7.5 IU HCG (Calbiochem). After administration of HCG,females were mated with B6D2F1 males.). Fertilized zygotes were isolated24 hours later. Zygotes were left in Hepes buffered CZB with 0.1% bovinetesticular hyaluronidase for several minutes at room temperature toremove any remaining cumulus cells. After washing, zygotes weretransferred to a new culture dish containing drops of bicarbonatebuffered CZB and placed at 37° overnight to obtain two-cell embryos.

Preparation of Tetraploid Embryos by Electrofusion

40 hours post HCG the blastomeres of two-cell embryos were electrofusedto produce one-cell tetraploid embryos. Electrofusion was carried out onin inverted microscope using the lid of a petri dish as amicro-manipulation chamber. Platinum wires were used as both electrodesand micromanipulators to align two cell embryos for fusion. A group of15 two-cell embryos was placed on the stage in a 200 ml drop of M2 media(Sigma). Embryos were aligned with the interface between their twoblastomeres perpendicular to the electrical field and a singleelectrical pulse of 100V with a duration of 100 ms was applied to eachindividually. Manipulation of a single group took less then fiveminutes. After electrofusion, embryos were returned to CZB media at 37°C. Embryos that had not undergone membrane fusion within 1 hour werediscarded.

Piezo Micromanipulator Injection of Tetraploid Blastocyts

For microinjection, 5-6 blastocysts were placed in a drop of DMEM with15% FCS under mineral oil. A flat tip microinjection-pipette with aninternal diameter of 12-15 um was used for ES cell injection. 15 EScells were picked up in the end of the injection pipette. The blastocystto be injected was held in the vicinity of the ICM with a standardholding pipette. The injection pipette, containing the ES cells waspressed against the zona opposite the inner cell mass. A brief pulse ofthe Piezo (Primatech Pmm, Ibaraki, Japan) was applied and the injectionneedle was simultaneously pushed through the zona and trophectodermlayer into the blastocoel cavity. The ES cells were then expelled fromthe injection pipette and pushed against the inner cell mass of theblastocyst. After injection of the entire group, blastocysts werereturned to CZB media and placed at 37° C. until transfer to recipientfemales.

Example 1

Assessment of the Effects of Genetic Heterogeneity of Donor Cells OnDevelopment of ES Cell-Tetraploid Pups.

The possible effect of genetic heterogeneity of the donor cells on thedevelopment of ES cell-tetraploid pups was tested by transferring inbredor F1 ES cells into tetraploid blastocysts and assessing survival.Injection of ES cells into the blastocoel cavity of tetraploidblastocysts was aided by the use of a piezo-driven micromanipulator andthe resulting composite embryos were transferred to recipient females.312 tetraploid blastocysts were injected with four different inbred EScell lines that gave rise to 20 pups (6%) that were alive and active atcesarean section. However, 17 of the 20 newborns died of respiratoryfailure within 30 minutes. Of the three remaining pups, two were unableto sustain respiration and died within the next few hours (Table 1).Only one inbred ES cell-tetraploid pup was able to sustain respirationand developed to adulthood. In contrast, of 344 tetraploid blastocystsinjected with 6 different F1 ES cell lines, 60 (18%) developed to birth,51 of which (85%) survived to adulthood (Table 2). Thus, geneticheterogeneity of the donor ES cells has a significant effect onlong-term survival of both nuclear clones and ES cell-tetraploid pups.

It has been previously shown that continued passage of ES cells isdetrimental to their developmental potency (Wang, Z. Q., et al., Mech.Dev., 62:137-145 (1997); Nagy, A. et al., Proc. Natl. Acad. Sci. USA,90:8424-8428 (1993)). In order to assess whether continuous in vitroculture would impair the survival of F1 ES cell-tetraploid pups, a129Sv×C57BL/6 ES cell line (V6.5) was kept continuously in culture andinjected into tetraploid blastocysts after prolonged passage. Noimpairment of postnatal survival of the resulting ES cell-tetraploidpups was noted after either 15 or 25 passages. In addition, F1 EScell-tetraploid mice were produced from cells that had been subjected totwo consecutive rounds of drug selection. First, selection withpuromycin was used for isolating cells that carried a targeted insertionof a tet-transactivator gene in the Rosa26 locus. Second, hygromycinselection was used to isolate cells with a tet-inducible promoterdriving expression of a hygromycin-thymidine kinase cassette in a randomlocus. Injection of these double-selected cells into 20 tetraploidblastocysts resulted in one full-term pup, which survived to adulthood(Table 2). The results described herein indicate that live, adult mice,entirely derived from ES cells can be generated from F1 ES cells evenafter long-term passage of the cells in culture or after consecutiverounds of drug selection.

Example 2

Histological Assessment of Lungs.

ES cell-tetraploid pups derived from inbred ES cells appeared to sufferfrom respiratory distress after delivery. Histological analysis of bothinbred and F1 completely ES cell derived neonates was carried out.Examination of the lungs from inbred ES cell-tetraploid pups revealedthat the alveoli were not inflated, while the lungs of newborns derivedfrom F1 ES cells were fully inflated. In addition, interstitial bleedingwas often seen in inbred ES cell derived mice. These observationssuggest that the failure to initiate breathing and/or sustain normalcirculation likely contributed to postnatal death of inbred EScell-tetraploid pups.

Results described herein demonstrate that genetic heterozygosity is acrucial parameter influencing postnatal survival of pups derived from EScells by tetraploid embryo complementation. Pups derived from inbred EScells die perinatally with a phenotype of respiratory failure. Incontrast, the great majority (80 to 85%) of pups derived from F1 EScells survived to adulthood. The observed respiratory phenotype appearsto be due to the inbred nature of the ES cell genome.

The possibility of deriving mice directly from ES cells without theproduction of a chimeric intermediate has great potential forfacilitating the generation of animals with multiple geneticalterations. In conventional approaches, targeted ES cells are injectedinto diploid blastocysts to generate chimeric founders. The derivationof transgenic mice carrying the desired mutant allele requiresout-crossing these chimeras with wild type mice. Thus, the generation ofcompound animals that combine multiple desired alleles or transgenes intheir genome entails time-consuming and expensive cycles of crossingmice derived from different chimeric founders. In contrast, the EScell-tetraploid technology in combination with F1 ES cells allowsassembling multiple genetic alterations in the same ES cell line byconsecutive gene targeting cycles in vitro prior to generating mutantanimals. The desired transgenic mice with numerous genetic alterationscan be derived in a single step by injecting the multiply targeted F1 EScells into tetraploid blastocysts. Finally, unlike nuclear cloningtechnology, which has proven both difficult to master and transfer fromlaboratory to laboratory, the ES cell-tetraploid technology is easilyadapted to any laboratory currently creating chimeric mice by ES cellblastocyst injection.

At present, the mechanisms that permit long-term survival of clones andES cell-tetraploid pups derived from F1 but not from inbred ES cells areunclear. Though it is generally assumed that “hybrid vigor” is animportant parameter in animal survival under various selectiveconditions, it is not apparent whether wide-ranging chromosomalheterozygosity or heterozygosity at only a few crucial modifier loci isrequired. Examining the potency of ES cells that have been derived frombackcrosses between F1 mice and their parental inbred strains mayclarify this question. TABLE 1 Survival of Inbred ES Cell-TetraploidPups Pups Pups respirating Pups alive at after surviving ES cell 4Nblasts term C-section to adulthood line genotype injected (% Inj) (%Alive) (% Alive) J1 129/Sv 120  9(7.5) 0 0 V18.6 129/Sv 48  5(10) 1(20)0 V26.2 C57BL/6 72  3(4) 1(33) 0 V39.7 BALB/c 72  3(4) 1(33) 1(33) TotalInbred 312 20(6) 3(15) 1(5)

TABLE 2 Survival of F1 ES Cell-Tetraploid Pups Pups Pups respiratingPups alive at after surviving ES cell 4N blasts term C-section toadulthood line Genotype injected (% Inj) (% Alive) (% Alive) V6.5C57BL/6X 72 18(25) 17(94) 16(89) 129/Sv V6.5* C57BL/6X 60 11(18)  9(81) 9(81) 129/Sv V6.5** C57BL/6X 20  1(15)  1(100)  1(100) 129/Sv 129B6129/Svx 48  2(4)  1(50)  1(50) C57BL/6 F1.2-3 129/Svx 48  4(8)  3(75) 3(75) M. Cast. V8.1 129/Svx 24  7(30)  7(100)  7(100) FVB V17.2 BALB/cX48 13(27) 12(92) 11(85) 129/Sv V30.11 C57BL/6X 24 4(30) 4(100) 3(75)BALB/c Total F1 344  60(18) 54(90) 51(85)*ES cell subclone targeted at the Rosa26 locus.**ES cell subclone serially targeted once at the Rosa26 locus and oncewith a random insertion.

Example 3

Generation of Fertile Mice Carrying A Mutation of Interest.

A male targeted F1 ES cell line was made as described above. The cellline was then screened by Southern blot for subclones that have lost theY chromosome. The probe used in the Southern blot to identify targeted(mutant) ES subclones which have lost the Y chromosome was a Ychromosome probe against repetitive elements. This probe was previouslydescribed by Lamar, E. E. and Palmer, E., Cell, 37:171-177 (1984).

Subclones that have lost the Y chromosome were taken and used to makemice by tetraploid embryo complementation as described above. The femaleES mice produced were shown to be fertile, indicating that they cantransmit the mutation of interest.

Male ES mice were produced from parent cell lines as described above.The male ES mice produced were shown to be fertile.

The loss of the Y chromosome, as shown by Southern blot analysis, is ageneral phenomenon in several ES lines, as demonstrated by the resultsshown in Table 3. Loss of the Y chromosome occurs at a frequency thatcan easily and routinely be screened for, as shown in Table 4. TABLE 3Frequency of Y Chromosome Loss in WT and Targeted ES Cell Lines # of #of subclones subclones lacking Y screened for repeats ES cell lineGenotype Passage # loss of Y (% of screened) V6.6 129svJaex P8-9 4488(1.8) c57B6 B6129 C57B6x P6 146 4(2.7) 129svJae J1 129svJae P7 2895(1.7) V6.5LJGF1 129svJaex 1X 210 3(1.4) preport c57B6 transfection

TABLE 4 Production of Male and Female ES-Tetraploid Mice from TargetedCell Lines Neonates 4N alive Mice Sex of surviving Blasts at C-SectionSurviving to mice ES cell line* Inj (% blasts) Adulthood Female Male FLPreporter 68 7(10.2) 3(4.4) 0 3 FLP reporter 63 7(11.1) 6(9.5) 6 0Subclone #66 FLP reporter 46 5(10.8) 5(10.8) 5 0 Subclone #315*The parent line (Flpreporter targeted line) generated male mice whilethe two subclones tested (#66 and #315), which were identified as havinglost the Y chromosome, generated female mice.

Both male (generated from the F1 preporter targeted line) and female(generated from subclone #315) mice carry the exact mutation at the samelocus in the genome and when intercrossed will directly lead tohomozygous mutant off-spring. Both male and female ES mice were shown tobe fertile.

While this invention has been particularly shown and described withreference to particular embodiments thereof, it will be understood bythose skilled in the art that various changes inform and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of identifying XO F1 ES cells, comprising screening apopulation of F1 ES cells for F1 ES cells that are XO F1 ES cells,wherein said population is a mixture of XO F1 ES cells and XY F1 EScells.
 2. The method of claim 1, wherein screening is carried out toidentify F1 ES cells in which spontaneous loss of the Y chromosome hasoccurred, thereby producing XO F1 ES cells.
 3. The method of claim 2,wherein said population is a population of wildtype F1 ES cells or apopulation of mutant F1 ES cells.
 4. The method of claim 3 wherein theF1 ES cells are mouse cells.
 5. The method of claim 3, wherein screeningis carried out using a Y chromosome probe against repetitive elements orPCR amplification.
 6. A method of isolating XO F1 ES cells from XY F1 EScells, comprising (a) screening a population of F1 ES cells for loss ofthe Y chromosome, wherein said population is a mixture of XO F1 ES cellsand XY F1 ES cells; and (b) isolating F1 ES cells lacking the Ychromosome, thereby isolating XO F1 ES cells.
 7. The method of claim 6,wherein said population is a population of wildtype F1 ES cells or apopulation of mutant F1 ES cells.
 8. The method of claim 7, wherein theF1 ES cells are mouse cells.
 9. The method of claim 7, wherein screeningis carried out using a Y chromosome probe against repetitive elements orPCR amplification.
 10. Canceled.
 11. A method of producing an XO femalenon-human mammal, comprising introducing XO F1 ES cells identified usingthe method of claim 1 into tetraploid blastocysts of the same mammalianspecies under conditions that result in production of an embryo andtransferring the resulting embryo into a foster mother which ismaintained under conditions that result in development of liveoffspring, thereby producing an XO female non-human mammal.
 12. Themethod of claim 11, wherein said XO F1 ES cells are wildtype XO F1 EScells or mutant XO F1 ES cells.
 13. The method of claim 11, wherein saidXO F1 ES cells are mouse cells and the non-human mammal is a mouse. 14.A non-human mammal produced by the method of claim
 11. 15. A non-humanmammal produced by the method of claim
 12. 16. A mouse produced by themethod of claim
 13. 17. A method of producing an XO female mouse,comprising introducing mouse XO F1 cells identified using the method ofclaim 1 into tetraploid mouse blastocysts under conditions that resultin production of an embryo and transferring the resulting embryo into afoster mother which is maintained under conditions that result indevelopment of live offspring, thereby producing an XO female mouse. 18.The method of claim 17, wherein the mouse XO F1 ES cells are wildtypemouse XO F1 ES cells or mouse mutant XO F1 ES cells.
 19. A mouseproduced by the method of claim
 17. 20. A mouse produced by the methodof claim
 18. 21. An XO female mouse produced by (a) introducing mouse XOF1 ES cells into tetraploid mouse blastocysts under conditions thatresult in production of an embryo, said mouse XO F1 ES cells identifiedby screening a population of mouse F1 ES cells for F1 ES cells that areXO F1 ES cells, wherein said population is a mixture of mouse XO F1 EScells and mouse XY F1 ES cells; and (b) transferring the resultingembryo into a foster mother which is maintained under conditions thatresult in development of live offspring, wherein the live offspring areXO female mice.
 22. The XO female mouse of claim 21, wherein screeningis carried out to identify F1 ES cells in which spontaneous loss of theY chromosome has occurred, thereby producing XO F1 ES cells.
 23. The XOfemale mouse of claim 22, wherein said population of mouse F1 ES cellsis a population of mouse wildtype F1 ES cells or a population of mousemutant F1 ES cells.
 24. The XO female mouse of claim 22, whereinscreening is carried out using a Y chromosome probe against repetitiveelements or PCR amplification.
 25. A method of isolating XO F1 ES cellsfrom XY F1 ES cells, comprising: (a) screening a population of F1 EScells for loss of the Y chromosome, wherein (i) said population is amixture of XO F1 ES cells and XY F1 ES cells; and (ii) said populationof F1 ES cells are generated by introducing into male F1 ES cells anegative selection marker under conditions appropriate for insertion ofthe negative selection marker in the Y chromosome of male F1 ES cells,thereby producing a mixture of F1 ES cells comprising male F1 ES cellsin which the negative selection marker is inserted in the Y chromosomeand other male F1 ES cells, some of which do not contain a Y chromosome;and subjecting the resulting F1 ES cells to conditions that result inthe death of male F1 ES cells in which the Y chromosome has the negativeselection marker inserted therein and do not result in the death of maleF1 ES cells that lack a Y chromosome; and (b) isolating F1 ES cellslacking the Y chromosome, thereby isolating XO F1 ES cells.
 26. Themethod of claim 25, wherein said population is a population of wildtypeF1 ES cells or a population of mutant F1 ES cells.
 27. The method ofclaim 26, wherein the F1 ES cells are mouse cells.
 28. The method ofclaim 26, wherein screening is carried out using a Y chromosome probeagainst repetitive elements or PCR amplification.
 29. A method ofproducing an XO female mouse, comprising injecting mouse XO F1 cellsinto tetraploid mouse blastocysts under conditions that result inproduction of an embryo and transferring the resulting embryo into apseudopregnant female mouse which is maintained under conditions thatresult in development of live offspring, thereby producing an XO femalemouse.
 30. A mouse produced by the method of claim
 29. 31. A method ofproducing a mutant XO female mouse, comprising injecting mutant mouse XOF1 cells into tetraploid mouse blastocysts under conditions that resultin production of an embryo and transferring the resulting embryo into apseudopregnant female mouse which is maintained under conditions thatresult in development of live offspring, thereby producing a mutant XOfemale mouse.
 32. A mouse produced by the method of claim
 31. 33. Amethod of producing a transgenic mouse homozygous for a transgenecomprising breeding an XO female mouse and a male mouse, wherein said XOfemale mouse and said male mouse are produced from the same non-inbredES cell clone comprising said transgene.
 34. A method of producing amouse, comprising introducing mouse non-inbred ES cells into mousetetraploid blastocysts under conditions that result in production of anembryo and transferring the resulting embryo into a pseudopregnantfemale mouse which is maintained under conditions that result indevelopment of live offspring, wherein 80 to 85% of said offspringsurvive to adulthood.
 35. An F2 mouse strain produced by breeding an XOfemale mouse and a male mouse, wherein said XO female mouse and saidmale mouse are produced from the same non-inbred ES cell clonecomprising said transgene.